Immunogenic compositions comprising progastrin and uses thereof

ABSTRACT

The present invention is drawn to immunotherapeutic methods to treat tumors/cancers that produce progastrin ectopically or are dependent on progastrin for their growth. Disclosed herein are immunogenic compositions comprising agents that target progastrin, agents that target the progastrin receptor, annexin II, or both. Such a composition may be administered in combination with chemotherapy or to an individual who had been previously subjected to chemotherapy or radiation therapy. The cancers that may be treated using such a composition may include but are not limited to colon cancer, breast cancer, lung cancer or pancreatic cancer.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 12/002,979,filed Dec. 19, 2007, now U.S. Pat. No. 7,854,932, issued Dec. 21, 2010,which claims the benefit of provisional application U.S. Ser. No.60/875,732, filed Dec. 19, 2006, the entirety of which is herebyincorporated by reference.

FEDERAL FUNDING LEGEND

This invention was produced using funds obtained through a NationalInstitutes of Health grant (R01 CA097959). Consequently, the Federalgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of immunology andvaccine development. More specifically, the present invention providesvarious immunogenic compositions comprising progastrin and their use inthe prevention and treatment of cancer.

2. Description of the Related Art

Colorectal cancers are one of the most common forms of cancers in menand women in the US and one of the leading causes of death. The sequenceof genetic events that are associated with the multi-step process ofdeveloping different types of familial colon cancers are well-known.Although advances in molecular genetics have led to better riskassessment and earlier diagnosis of colorectal cancer, it still remainsa deadly disease for majority of the patients due to lack of effectiveadjuvant treatments. Such adjuvant or systemic treatments are likely toarise from a better understanding of factors that regulate proliferationof colonocytes and colon cancer cells. Just as genetic instability dueto the inheritance of specific genetic defects plays a dominant role ininitiation and progression of familial cancers, hyperproliferation islikely to play a permissive role in the initiation and progression ofsporadic cancers. Hence, hyperproliferation is recognized as a riskfactor that can initiate dysplastic growth, resulting in accumulation ofgenetic defects and progression to colon cancer. Gastrins represent agroup of growth factors that can potentially play a prominent role inproliferation of normal and cancerous intestinal cells.

The gastrin gene is normally expressed and processed in the brain and inthe antral stomach of mammalian species. The full length progastrin (PG)peptide, which is 80 amino acids, undergoes enzymatic deletions both atthe C and N terminal ends, to finally generate the fully processedC-terminally amidated gastrin peptides in the neuro endocrine cells(FIG. 1A). G17 and G34 amino acid gastrin peptides stimulate acidsecretion and growth of the gastrointestinal (GI) tract. Although thecolon cancer cells express the gastrin gene, they do not process theprogastrin peptides (1-2). Thus, patients with colorectal cancers (CRCs)are positive for significant levels of progastrin-like peptides in thecirculation. Some studies have reported the presence of elevated levelsof progastrin peptides but not gly-extended gastrin or gastrins inpatients with colorectal cancers (3).

Additionally, a significant percentage of human colon cancer (HCC) cellshave also been shown to require the expression of progastrin-likepeptides for maintaining the in vitro and in vivo growth of the cells(4). Downregulation of the gastrin gene resulted in the attenuation ofthe growth of gastrin dependent human colon cancer cells in vitro and invivo (4). A processing intermediate, gly-extended gastrin (GG) wasreported to exert potent growth factor effects on several target cellsincluding normal and cancerous intestinal epithelial cells (IEC) (5-6).Additionally, U.S. Pat. Nos. 5,786,213 and 6,165,990 disclose genetherapy of colorectal cancers using the anti-sense technology. However,the delivery of the anti-sense plasmids has not advanced significantlyand has remained a concern along with associated side effects.

The use of immunotherapy, on the other hand, has advanced significantlyand is currently being used for treatment purposes of many cancers bytargeting other cancer-related molecules such as EGF receptors, HER-2Neu Oncogene, Anti CD52, anti VEGF, Anti CD22, Anti CD80 etc. (7). Avaccine against the gastrin peptide (G17DT) was developed and has beenused in clinical trials with ambiguous results (8). The G17DT vaccine isa chemical conjugate of 9 N-terminal amino acids of G17 sequenceconjugated to DT (Diptheria Toxin). Since it is difficult to have auniform conjugate with similar composition and stoichiometric ratios ofpeptide to carrier, the immune response would vary from batch to batchof vaccine. Additionally, peptide vaccines produce limited immuneresponse. Another problem with the DT vaccine is that it may produceimmunosuppression against the DT-peptide sequences as most of the humansare immunized against Diptheria. Furthermore, the quality control andquality assurance of conjugate vaccine is difficult.

It is known that several receptor (R) sub-types mediate the biologicalfunctions of gastrin-like peptides and progastrin-like peptides. Ofthese, CCK2R and its splice variants mediate biological effects ofprimarily CCK and gastrin-like peptides. On the other hand, novelproteins such as Annexin II mediate growth factor effects ofprogastrin-like peptides (9). The G17 N-terminal based vaccineessentially targets G17 and thus inhibits binding to CCK2R; progastrinbased vaccine, on the other hand, may target the actions of progastrinpeptides and inhibit binding to receptors such as Annexin-II. This mayexplain the high efficacy of the current vaccine.

Thus, prior art is deficient in an immunogenic composition that can beuseful as vaccine in the treatment of cancers that produce progastrinectopically or are dependent on progastrin for their growth. The currentinvention fulfils this long standing need in the art.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there is provided acomposition comprising an agent targeting progastrin or an agenttargeting annexin II or both of the agents. The agent comprisesprogastrin or a fragment thereof, an antibody directed against theprogastrin or the fragment thereof or the progastrin or specific siRNAtargeting gastrin gene. The agent targeting annexin II comprises anantibody directed against annexin II, annexin II specific siRNA orannexin II specific antisense oligonucleotide. The agents describedherein may be formulated in a pharmaceutically acceptable adjuvant, adelivery system or a combination thereof.

In a related embodiment of the present invention, there is provided amethod of inhibiting proliferation of a neoplastic cell. This methodcomprises contacting the neoplastic cell with the composition discussedsupra. The composition may comprise either an agent that targetsprogastrin or an agent that targets annexin II or both. Such a contactinhibits the growth-inducing activity of progastrin in the cell, therebyinhibiting the proliferation of the neoplastic cell.

In another related embodiment of the present invention, there isprovided a method of treating a cancer in an individual. This methodcomprises administering a pharmacologically effective amount of thecomposition discussed supra. The composition may comprise either anagent that targets progastrin or an agent that targets annexin II orboth. Such administration inhibits gastrin-induced proliferation ofcancer cell, thereby treating the cancer in the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings have been included herein so that theabove-recited features, advantages and objects of the invention willbecome clear and can be understood in detail. These drawings form a partof the specification. It is to be noted, however, that the appendeddrawings illustrate preferred embodiments of the invention and shouldnot be considered to limit the scope of the invention.

FIGS. 1A-1B are schematic representations of gastrin. FIG. 1A showsprocessing of gastrins. FIG. 1B shows peptide structure of G17 inrelation to that of recombinant human progastrin. The recombinantpurified hPG (rhPG) was detected by Western immunoblot Analysis usingspecific anti-hPG-antibodies (Abs). The 21 kDa band represents thefusion protein (FP) used for expressing rhPG in an E. coli system. Themolecular markers co-run in Lane 1. Specificity of antibodies fordetecting G17 and hPG was also examined. Gastrin-like peptides (G17,glycine-extended gastrin (GG), cholecystokinin (CCK-8) and hPG) weredetected in a slot blot assay using anti-G17-Abs or anti-hPG Abs.Anti-G17-Ab was specific for G17 and did not detect other gastrin-likepeptides to any significant extent. Similarly, anti-hPG-Ab was specificfor progastrin-like peptides and did not detect any other gastrin-likepeptide.

FIGS. 2A-2D shows relative levels of gastrin RNA in freshly obtained(non-frozen) hyperplastic polyps and adenomatous polyps compared to thatin normal mucosa of the colon. The data is presented herein as a scatterplot wherein each value represents the relative levels of gastrin RNA ineither each patient sample (FIGS. 2A-2C) or from all of the patients inthat group as mean±SE (FIG. 2D) compared to that in an arbitrarilychosen control normal mucosal sample.

FIGS. 3A-3B show immunohistochemical staining of tumors from patientswith adenocarcinoma. FIG. 3A shows immunohistochemical staining oftumors from patients with adenocarcinomas with an antibody directedagainst progastrin (PG-Ab). Sections of adenocarcinomas andhyperplastic/dysplastic colonic crypts from several patients wasanalyzed for the presence of progastrin peptide, histochemically withthe progastrin-Ab. Adjacent normal colonic crypt samples from the samepatient were also stained with progastrin-Ab. Representative data fromone patient is shown. Normal colonic crypts were negative for stainingwhile several dysplastic crypts stained significantly. Almost alladenocarcinomas were heavily stained. FIG. 3B shows immunofluorescentstaining of representative human patient samples from adenomatous polypsand adenocarcinomas with either PG-antibodies or Annexin II-antibodies.The staining of PG-antibodies and Annexin II-antibodies was detected byusing secondary antibodies that were tagged with either Texas Red orFITC-Green fluorescent dyes, as shown. In normal colonic (N. Colon) andAdenoma samples from patients, PG was detected with FITC green labeledsecond antibody, and Annexin-II staining was detected with Texas redlabeled second antibody. But in adenocarcinoma samples from patients, PGstaining was detected by Texas red tagged antibody and Annexin-IIstaining was detected by FITC-Green labeled antibody. The in situbinding of PG with Annexin II was established in the merged imagesshowing yellow staining (FIG. 3B). The relative levels of progastrin andAnnexin II and the relative co-localization (binding) of PG with ANX-IIin 4-5 patient samples from normal mucosa, adenomas and adenocarcinomasare presented in Table II.

FIG. 4A-4N show that PG-R (Annexin II) plays an important role in theprogastrin gene dependent cancers. FIG. 4A shows a hypothetical modeldepicting relative binding of progastrin (PG), G17 and CCK8 to CCK2-Rand PG-R (Annexin II) binding sites and resultant biological effects,based on published findings from our group as described elsewhere. FIGS.4B-4C show in vitro binding of Annexin II with PG peptides, whereincubation of recombinant Annexin II and progastrin, in vitro, resultedin co-immunoprecipitation of the ligand and its receptor, as detected inWestern Blots (FIG. 4B) and by coomassie blue (FIG. 4C). FIGS. 4D-4Gshow in situ binding of Annexin II with progastrin in colon cancercells, expressing progastrin peptide (HCT-116 cells). FIG. 4D shows thepresence of progastrin binding proteins of molecular mass of ˜80 KDa,(band a), 40-50 KDa (band b) and ˜36 KDa (band c). The proteins in bandc were identified as annexin-II as shown in FIG. 4E. Using pull-downassays in FIG. 4F with progastrin-antibody, Annexin IIco-immunoprecipitated with progastrin from the cellular lysates of arepresentative colon cancer cell line, HCT-116, and was detected byWestern Blot analysis. Using immunofluorescence in FIG. 4G, progastrinwas found to co-localize very strongly with Annexin II in HCT-116 cells,in situ, confirming a strong binding of PG with Annexin II in situ. Inthis figure, red flourescense depicts PG, green fluoroscense depictsannexin II and blue depicts nuclei stained with DAP1. Merged yellowimage depicts co-localization of PG and annexin-II in HCT-116 cellsexpressing autocrine PG. FIGS. 4H-4I shows that PG strongly co-localizedwith Annexin II in the proximal (P) colonic crypts of transgenic (Tg)mice (Fabp-PG) over-expressing progastrin in the colonic crypts. In FIG.4H, magnification of 20× was used, while in FIG. 4I, magnification of40× was used. Absence of PG expression in the distal (D) (data notshown) and proximal (P) colonic crypts of wildtype mice (WT) wasconfirmed. In FIG. 4I the upper panel depicts distal crypts from Tg miceand the lower panel depicts merged images from proximal crypts of Tgmice. The data in FIGS. 4H-4I thus confirmed that Annexin II functionsas an avid binding partner for progastrin peptides in vitro and in vivo.FIGS. 4J-4K show a critical need for Annexin II expression for measuringgrowth effects of progastrin on intestinal epithelial cells (i and ii)and colon cancer cells (9). Anti-sense clones of IEC-18 cells (FIG. 4J),down-regulated for ANX-II expression, were non-responsive to progastrin(PG), compared to sense clones (FIG. 4K). However, both the sense andanti-sense clones were equally responsive to fetal calf serum (FCS).Similarly, HCT-116 cells down regulated for autocrine expression ofannexin II, demonstrated a significant loss in the growth of theanti-sense clones in response to autocrine PG, compared to wildtype andsense clones (9). Treatment of intestinal epithelial cells (IEC-18)(FIG. 4L) and pancreatic cancer cells, (AR42J) (FIG. 4M) with Annexin IIantibodies, but not CCK₂R antibodies, resulted in the attenuation ofgrowth effects of progastrin, but not the growth effects of FCS. FIG. 4Nshows that Annexin II plays a critical role in the internalization ofprogastrin in the intestinal epithelial cells. Down-regulation ofAnnexin II expression using specific Annexin II siRNA, resulted in theloss of binding and internalization of progastrin in the IEC cells.Control, non-specific siRNA treatment had no effect. However,significant binding of annexin II was observed with PG, resulting ininternalization of the receptor and ligand (Yellow merged image), within10 minutes of PG treatment.

FIGS. 5A-5B show effect of antibodies on growth of HCT-116 cells inculture. Growth of cells was examined by an MTT assay. In FIG. 5A,growth of cells in response to 1% FCS was arbitrarily assigned a 0level. A decrease or increase in growth was recorded as % changecompared to levels seen in control (1% FCS stimulated) cells. FIG. 5Bshows effect of increasing concentration of anti-PG-Ab. 1:500 dilutionof anti-PG-Ab completely reduced the growth of FCS-stimulated levels tonon-stimulated levels.

FIGS. 6A-6C show immune response induced by hPG-fusion protein (FP)immunogen in different mouse models. FIG. 6A shows PG-Ab titers inBalb/c mice that were immunized with hPG-fusion protein (FP) immunogenat the indicated time points. The immunized Balb/c mice were inoculatedwith mouse CA cells at week 10 and sacrificed at week 14 and the tumorsin the mice were weighed. Control mice received adjuvant alone. Theweight of the tumors is indicated by an arrow against Ab titer of eachmouse in mgs. Tumor weights from 3 of 6 animals are presented. Theweights were ˜1530% lower than that in control mice (820-1000 mg). Themouse that demonstrated highest titers between weeks 8-12 had thesmallest tumor which was necrotic compared to solid tumors in othermice. FIGS. 6B-6C show effect of vaccination with rhPG-FP immunogen oncolon carcinogenesis in FVB/N mice. FVB/N mice were immunized with thehPG-fusion protein (FP) immunogen by 3-4 weekly injections and the PG-Abtiters were measured as described for the Balb/c mice. Once the micedeveloped significant PG-Ab titers, the mice were treated withazoxymethane (AOM) and the total number and size of tumors measuredafter 6 months of AOM treatment. The number of tumors (FIG. 6B) and thetumor burden (FIG. 6C) is presented herein. *=p<0.05 vs control values.Control mice were immunized with adjuvant alone.

FIGS. 7A-7E show effect of hPG fusion-protein (FP) on coloncarcinogenesis in transgenic PS21 Fabp-PG mice. FIG. 7A shows the totalnumber of aberrant crypt foci (ACF) in either PS21 transgenic mice(over-expressing PG in the colon) or wild type (WT) mice as Mean±SDvalues (n=10 mice/group) from a representative experiment. FIG. 7B showsthe % change in the number of ACF/mouse colon in PS21 transgenic mice ingroups that were vaccinated with either thioredoxin (Thio) alone, orfusion protein (FP) containing thioredoxin plus progastrin, orprogastrin (PG) alone, compared to that in the corresponding WT mice,wherein the levels in WT mice were arbitrarily assigned 100% values.FIG. 7C shows anti-progastrin antibody titers in wild type and PS21(Transgenic) mice after immunization with recombinant proteins:thioredoxin (Thio), thioredoxin-progastrin fusion protein (FP) andprogastrin (PG). FIG. 7D shows anti-progastrin titers in transgenic mice(PS21) mice expressing human progastrin after immunization withrecombinant proteins: Thioredoxin, Thioredoxin-progastrin fusion proteinand progastrin. FIG. 7E shows anti-thioredoxin titers in wild type andPS 21 mice.

FIGS. 8A-8C show binding of ¹²⁵I-rhPG and ¹²⁵I-BH-CCK8 to a pancreaticcancer cell line, AR42J and intestinal epithelial cells (IEC). Cellswere incubated with the indicated radio labeled ligand in the presenceor absence of increasing concentrations of either CCK8, G17 or rhPG. Therelative binding affinity of the gastrin like peptides for the specificbinding sites for rhPG (FIGS. 8A-8B) (Annexin II) and CCK8 (FIG. 8C) toCCK₂R was calculated. Data in each point represents the mean values oftriplicate measurements from a single experiment and is representativeof 2 similar experiments. The intra-experimental variation for each datapoint was <5-10%. The nM concentration of each peptide used is presentedin a log-scale on the x-axis; the excess unlabeled peptide used fordisplacing the binding of the radio labeled ligand is presented inparentheses. The % loss in the relative binding of the radio labeledligand in the presence of the increasing concentrations of the indicatednon-labeled peptide is presented on the y-axis. IEC cells lack CCK₂Rbinding sites and therefore binding with radio-labeled CCK8 was notexamined with these cells.

FIGS. 9A-9D show relative levels of human gastrin RNA and hCCK2R RNA.FIG. 9A shows relative levels of human gastrin RNA in frozen (discarded)samples of adenocarcinomas and surrounding normal mucosal tissues(obtained from a tissue bank at UTMB). The relative levels of gastrinRNA, on an average as measured in each sample or in all samples(mean±SD) in that group are shown compared to that in an arbitrarilychosen normal control mucosal sample. FIGS. 9B-9C show relative levelsof hCCK2R RNA in freshly obtained (non-frozen) hyperplastic polyps andadenomas compared to that in the surrounding normal mucosa. The levelsare presented in relation to that measured in an arbitrarily chosennormal colonic mucosal control sample. The values represent an averageof triplicate measurements from a single sample (FIG. 9B) or mean±SD ofthe indicated number of samples/group (FIG. 9C). FIG. 9D shows relativelevels of hCCK2R RNA in frozen (discarded) samples of adenocarcinomasand surrounding normal mucosal tissues, obtained from the tissue bank atUTMB. The values presented herein is either a mean of triplicatemeasurements from a single specimen or a mean±SD of the indicated numberof specimens/group. The relative levels of CCK2R RNA in each sample ispresented as a ratio of that measured in an arbitrarily chosen controlnormal mucosal sample.

FIGS. 10A-10G show diagrammatic representation or sequences of thevectors or nucleic acid sequences encoding proteins or amino acidsequences of the proteins discussed herein. FIG. 10A is a diagrammaticrepresentation of the expression vector (pET32), that was used forexpressing hgastrin cDNA. The codons for hgastrin cDNA was optimized forexpression in E. coli and cloned in pET32 at the NspV-HINDIIIrestriction sites. In FIG. 10B shows details of the pET32 vector. Thisvector was designed for cloning and high level expression of peptidesequences fused with the 109 amino acid thioredoxin, TRN.TAG™, protein.Cloning sites are available on this vector for producing fusion proteinswhich contain the cleavable His.tag sequences for detection andpurification. FIGS. 10C-10F show the blast sequence of hgastrin cDNA,used for cloning into the expression plasmid. The nucleotide sequencefor the native coding cDNA for human gastrin gene (and hence, PG) isshown in comparison to the sequence that was used after codonoptimization. FIG. 10G shows the amino acid sequence of the progastrinpeptide thus expressed by the recombinant expression vector.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses immunogenic compositions comprisingprogastrin or a fragment thereof, a progastrin antibody, a progastrinsiRNA, an annexin II antibody, an annexin II siRNA and their use in thetreatment of cancers expressing the gastrin gene and the progastrin-likepeptide. Progastrin and gastrin peptides are expressed in neuroendocrinecells in the brain and the antral part of the stomachs and processedinto precursor peptide and glycine extended form of gastrins (GG).Additionally, subsequent amidation at the C-terminal end generatesamidated forms of gastrins (G17, G24). Under physiological conditions,only processed forms of gastrins (G17, G34) are present as majorcirculating forms of gastrins (FIG. 1A).

The peptide structure of G17 and PG is diagrammatically presented inFIG. 1B. All antibodies derived against the C-terminal end of thepeptides demonstrated specificity for the class of gastrin peptidessince different forms of gastrin differ significantly from each other atthe C-terminal end as shown in the western blot data in bottom righthand side in FIG. 1B. The anti-PG-Ab was very effective in detecting the9 kDa molecule. Similarly dot blot assay in FIG. 1B shows thatanti-PG-Ab specifically detected the presence of PG molecules but didnot detect the presence of any other gastrin-like peptide. Theanti-G17-Abs similalry were very specific for detecting amidatedgastrins like G17, but did not detect GGly or rhPG-like molecules anddid not detect a closely related amidated peptide, CCK-8. Theseantibodies can be used for immunohistochemical analysis, as disclosedherein, for measuring PG and G17 in the serum by either RIA or westernblot analysis.

The present invention also discloses that the gastrin gene was expressedat a very early stage during colon carcinogenesis in humans and thateven hyperplastic polyps and adenomas express the gastrin gene and thePG-like peptides (FIGS. 2A-2D; Table 1).

TABLE 1 Gastrin gene expression in polyps relative to that in HCT- 116cells in relation to locus and pathology of polyps Count Mean MedianStd. Dev Max Locus Proximal 15 664.6512 0.136 2378.397 9247.3 Transverse5 15948.96 38.7 35604.38 79640.0 Distal 1 5.36 5.36 5.36 Rectum 4 0.50340.327 0.5371101 1.25 Pathology Hyperplastic 10 4.84706 0.1215 12.0397138.7 Microadenoma 1 5.36 5.36 5.36 Adenoma 14 6436.636 7.695 21210.5479640.0 Adenocarcinoma 1 0.54 0.54 0.54

Furthermore, staining of human adenomas and adenocarcinomas withanti-progastrin antibodies (FIGS. 3A-3B) corroborated the findings inFIGS. 2A-2D. A full length recombinant human PG peptide (rhPG) which wasbioactive was generated recently and demonstrated potent growthfactor-like effects of recombinant human PG peptide on intestinalepithelial cells (IEC), colon cancer cells and pancreatic cancer cells(9-13). The full length progastrin peptides are extended at both theN-terminal and C-terminal ends of the gastrin peptide (FIGS. 1A-1B),which contributes to a significant difference in the receptor subtypesthat bind PG peptides vs the gastrin peptides. Additionally, it is knownthat several receptor (R) sub-types mediate the biological functions ofgastrin-like peptides and PG-like peptides. Of these, CCK2R and itssplice variants mediate biological effects of primarily CCK andgastrin-like peptides. On the other hand, novel proteins such as AnnexinII bind PG with high affinity (FIGS. 4B-4I), and mediate growth factoreffects of PG-like peptides on colon cancer cells, intestinal epithelialcells and pancreatic cancer cells (FIGS. 4J-4M) (9, 13-14).

It has also been suggested that gastrin-like peptides can mediate bothstimulatory or inhibitory effects on the growth of target cells viaCCK₂R. PG peptides, however, do not bind CCK2R and mediate onlystimulatory effects on the growth of intestinal epithelial cells (IEC)and human colon cancer cells (HCC) via receptors other than CCK2R (9,13-14). Furthermore, studies with transgenic mice demonstrated that miceoverexpressing PG and PG-like peptides were at an increased risk forcolon carcinogenesis in response to chemical carcinogens (azoxymethane,AOM), while mice overexpressing gastrins were at reduced risk for coloncarcinogenesis (15-17). It was also suggested that while the presence ofcirculating PG-like peptides may function as a co-carcinogen andincrease the risk for initiation of colon carcinogenesis, the presenceof autocrine PG may further enhance the growth of the colorectal cancertumors and progression of the disease. Thus, both circulating andautocrine progastrins can potentially play an important role ininitiation and progression of colon cancer.

In summary, it was observed that, 1) gastrin peptides decrease ratherthan increase the risk of colon carcinogenesis, 2) both endocrine andautocrine progastrins increase the risk of colon carcinogenesis, and 3)colorectal cancer mainly express gastrin gene product, progastrin (FIGS.2A-2D, 3A-3B, 4A-4N, 5A-5B), and the receptor Annexin II (FIG. 3B), butdo not express amidated gastrins (FIGS. 5A-5B) and CCK2R (FIGS. 9B-9C).Therefore, targeting progastrin and/or its receptor (Annexin II) ratherthan gastrins can be expected to be significantly more effective ininhibiting the growth of gastrin gene dependent colorectal tumors. Ahypothetical model depicting relative binding of progastrin, G17 andCCK8 and PG-R (Annexin-II) binding sites and resultant biologicaleffects is shown in FIG. 4A. Thus, differential expression of CCK2R andPG-R (Annexin II) on colonic mucosal cells may dictate co-carcinogenicvs protective effects of G17. Circulating progastrin was shown topromote proliferation and was co-carcinogenic on colonic mucosa intransgenic mice over-expressing hPG (hGAS and FABP-hGAS mice), butelevated levels of G17 in transgenic mice (INS-GAS) reduced thecarcinogenic effects of AOM (15-16, 18). The present inventiondemonstrates that antibodies against PG (FIGS. 5A-5B) and/or Annexin II(FIGS. 4J-4M) were most effective in attenuating the growth of gastringene-dependent human colon cancers in vitro whereas antibodies againstgastrin peptides were completely ineffective (FIGS. 5A-5B). Thisprovides a strong evidence for potent growth promoting andco-carcinogenic role of autocrine progastrin on growth andtumorigenicity of gastrin gene-dependent colon cancer cells. Thus,developing auto-antibodies against progastrin-like peptides and/orAnnexin II will reduce the risk of colon carcinogenesis and growth ofcolon cancer tumors in vivo.

A full length progastrin protein fused with a carrier protein,Thioredoxin (from bacteria) was examined herein as an effective vaccineagainst colon carcinogenesis in three animal models (Balb/C, FVB/N,FABP-PG). In these mouse models, the immunogen generated a strongresponse and very high titers of specific anti-hPG-Abs were measuredwithin 4-8 weeks of vaccination. In the Balb/c mouse model, thepotential effectiveness of the antigenic protein as a vaccine againstthe growth of a gastrin dependent mouse colon cancer cell line (CA) wasobserved (FIG. 6A). Mouse colon cancer cells are derived from Balb/ctumors and grow in Balb/C mice. Mouse PG shares ˜80% homology at theC-terminal end and <65% homology at the N-terminal end with hPG. Coloncancer tumor weights in mice treated with hPG-immunogen were reduced15-30% compared to that in control mice. Additionally, in one mouse thatdemonstrated the highest antibody titer, tumors were not only thesmallest but also necrotic (FIG. 6A).

In the other mouse model, FVB/N mice were immunized against hPG-FP andtreated with AOM to examine if anti-PG-Abs reduce the risk of coloncarcinogenesis in these mice. The total number of tumors (FIG. 6B) andtumor burden/mouse (FIG. 6C) were significantly reduced (p<0.05) in miceimmunized against hPG peptide compared to mice immunized with theadjuvant alone. In a third mouse model, FABP-PG where full length humanprogastrin would be an auto antigen in a transgenic mouse modelexpressing full length human progastrin, the fusion protein was used totest the ability of vaccine to elicit autologous immune response againstprogastrin. The progastrin fusion protein was able to elicit strongresponse in the transgenic as well as wild type mice. Progastrin alonewas much less effective while thioredoxin was not effective at all (FIG.7C). These results demonstrated that progastrin containing fusionprotein was an effective vaccine against colon carcinogenesis inresponse to AOM.

Both tumorigenesis (FIGS. 6B-6C) and pre-neoplastic markers ofcarcinogenesis, aberrant crypt foci, (FIGS. 7A-7B) were significantlyreduced in mice vaccinated with fusion progastrin protein, while micevaccinated with either progastrin or thioredoxin alone showed nodecrease in carcinogenesis. Aberrant crypt foci are reliable earlymarkers of colon carcinogenesis and are used to predict tumorigenesis inrodent models and human patients (19-20). Thus, the results discussedsupra provide strong evidence that the fusion protein could be used asan effective vaccine against either the initiation of coloncarcinogenesis in patients positive for high levels of progastrin or canbe used to significantly reduce the growth of gastrin-dependentcancers/tumors in patients.

It was demonstrated herein that relative levels of gastrin RNA wassignificantly lower in normal mucosa tissue than in frozen specimens ofadenocarcinomas (FIG. 9A). Additionally, low to negligible levels ofCCK2R RNA were detected in normal mucosa of patients from whom polypswere obtained. None of the polyps and adenomas expressed detectablelevels of CCK2R (FIGS. 9B-9C). Furthermore, in the frozen adenocarcinomasamples and in the majority of surrounding normal colonic mucosalsamples from these patients, no CCK2R RNA was measured (FIG. 9D). Onlyone normal mucosal sample was positive for CCK2R RNA. Thus, while thelevels of gastrin RNA were higher in adenocarcinoma, the levels of CCK₂RRNA were lower in such samples. This presence of lower levels of CCK₂Rin the normal colonic mucosa and the almost complete absence of CCK₂Rexpression in the colonic tumors suggests that CCK₂R does not play animportant role in colon carcinogenesis. On the other hand, the highaffinity receptors for progastrin, Annexin II, was increasinglyexpressed in colonic tumors in the order ofadenocarcinomas>adenomas>normal colonic mucosa (FIG. 3B, Table II),further accentuating an important role of Annexin II and progastrin incolon carcinogenesis.

TABLE 2 Relative intensity of PG/ANXII staining and co-localization innormal colonic mucosa (N) and corresponding Ad and AdCA N Ad N AdCASample PG ANXII PG ANXII PG ANXII PG ANXII 1 0 0 0 0(0)^(a) 0 1 01(0)^(a) 2 0 0 0 1(0) 0 1 0 2(0) 3 0 1 2 1(1) 0 1 3 4(3) 4 0 1 3* 3(3) 02 4 4(4) 5 — — 0 2(0) 0 3 5* 5(5) (Scale of staining: 0-5 = low-high;^(a)= relative colocalization of PG and ANXII (yellow) is given inparentheses. *= samples shown in FIG. 3B.

The E. coli vector containing the cDNA sequence for thioredoxin,upstream of hGastrin cDNA is shown in FIGS. 10A-10B. Additionally, FIGS.10C-10F show the changes made in the hGastrin sequence to obtainefficient expression of progastrin peptide. The complete 80-amino acidsequence of the expressed hPG peptide from this vector is shown in FIG.10G (SEQ ID NO: 3).

An etiological and prognostic role of gastrin gene/progastrin expressionin relation to known genetic alterations in colon cancers (such as lossof APC and p53 functions and gain of c-myc, cyclin D1, β-catenin andK-ras oncogenic functions) is established to develop effective protocolsfor preventative and treatment purposes (14). Differences in theexpression of the gastrin gene in different types ofhyperplastic/adenomatous growths and at different stages duringadenoma-carcinoma sequence is being determined. Additionally, the levelsof gastrin gene expression in tumors and serum progastrins are beingcorrelated with the clinical status of the patients as a means ofassessing prognostic value of measurements. Furthermore, in order toobtain reliable clinical data, the method of retrieval and storage ofthe specimens is examined to determine if the relative levels of gastringene expression (RNA and protein) are proportional to the serumprogastrin levels. It is contemplated that the combined use of theprogastrin containing fusion protein as a vaccine either alone or incombination with methods to down-regulate Annexin II, along with wellestablished chemotherapeutic protocols will not only provide much highertreatment rates in patients with PG-expressing cancers but also improvethe survival rates in these patients. Furthermore, since the gastringene and progastrin peptides are expressed in other cancers such aslung, breast, ovarian and pancreatic cancers besides colorectal cancers,the immunogenic compositions discussed herein may be used in treatingthe cancers that express the gastrin gene and are perhaps dependent ongastrin gene products (mainly progastrin) for their continued growth.

In summary, the immunogenic composition discussed herein may compriseprogastrin or N-terminal or C-terminal fragments of progastrin fusedwith a non-human protein and formulated with an adjuvant and/or deliverysystems. The progastrin may be a peptide comprising modified orunmodified amino acid sequences, recombinant progastrin or a fusionprotein of human Progastrin and a non-human protein which includes butis not limited to thioredoxin, tetanus toxoid, diphtheria toxin, AraC,TrpR or Psts. The fusion protein disclosed herein may further haveseveral variations which include but are not limited to a fusion proteinof human Progastrin fused with non human proteins where multiple copiesof Progastrin in tandem are fused with one or more nonhuman proteins, afusion protein of human Progastrin fused with non human proteins wheremultiple copies of Progastrin in tandem are fused with one or morenonhuman proteins and has a tag at N terminal or C terminal such as hexahistidine, GST etc. or a progastrin fusion protein that is formulatedwith an adjuvant such as CFA, IFA, Rib, emulsion, MDPs, apolysaccharide, lipopolysaccharide DNA like CpGs sequences, liposomes,sustained delivery system such as PLGs, hydrogels like chitosanalginates, chitosan metal chelates etc. Furthermore, the immunogeniccomposition disclosed herein may be used to generate antibodies whichcan then be administered to the individual. It is contemplated hereinthat the instantly claimed immunogenic compositions are superior to theG17DT vaccine that is known in the art since the instantly claimedimmunogenic compositions can be purified to a very high degree. Thecancers that can be treated using the immunogenic composition disclosedherein include but are not limited to colon cancer, pancreatic cancer,lung cancer, ovarian cancer and breast cancer. Furthermore, theimmunogenic compositions disclosed herein may be used in combinationwith Annexin II antibodies/siRNA and/or chemotherapy or in patients whohave been previously treated with chemotherapy or radiation therapy.

The present invention is directed to a composition, comprising an agenttargeting progastrin, where the agent comprises progastrin or a fragmentthereof, an antibody directed against the progastrin or the fragmentthereof or specific siRNA against progastrin, a pharmaceuticallyacceptable adjuvant, a delivery system or a combination thereof; or anagent targeting annexin II, where the agent comprises an antibodydirected against annexin II, annexin II specific siRNA, annexin IIspecific antisense oligonucleotide, a pharmaceutically acceptableadjuvant, a delivery system or a combination thereof; or both of theagents. Since formulating such a composition is routine in the art, theagents may or may not be formulated in a pharmaceutically acceptableadjuvant or a delivery system.

The progastrin or the fragment thereof in such a composition may be apeptide, a recombinant protein or a fusion protein. Further, the peptidemay comprise modified or unmodified amino acid residues of progastrin.The modified amino acids in the peptide may comprise alanine instead orarginine at position 36, 37, 73 and/or 74 and/or alanine instead oflysine at positions 53 and/or 54. Alternatively, the recombinant proteinin such composition may be produced in E. coli, yeast, salmonella, BCG,vaccinia or other expression system.

Furthermore, the fusion protein in such composition may comprisemultiple copies of progastrin in tandem fused with one or more non-humanproteins or multiple copies of progastrin in tandem fused with one ormore nonhuman proteins and tagged at N-terminal or C-terminal. Thenon-human protein in such fusion protein may include but is not limitedto a protein from E. coli, yeast, streptococcus or other eukaryotes. Theprotein from other eukaryotes includes but is not limited to TetanusToxoid, Diphtheria toxin, AraC, TrpR or Psts. Additionally, the tag atthe N or C-terminal is not limited to but may include hexa histidine,GST, cellulose binding protein, chitin binding protein, protein-A,protein-G or dihydrofolate reductase.

Examples of the adjuvant include but is not limited to CFA, IFA, Rib,emulsion, MDPs, a polysaccharide or a lipopolysaccharide DNA. Thelipopolysaccharide DNA may be a CpG sequence. Additionally, the deliverysystem includes but is not limited to a nanoparticle, liposome, PLG orhydrogel. Examples of the hydrogel include but is not limited tochitosan alginates or chitosan metal chelates. Furthermore, thecomposition may be a lyophilised powder that can be reconstituted, asuspension or an emulsion. Examples of routes of administering thecomposition discussed herein may include but is not limited tointramuscular, subcutaneous, intravenous, intranasal, oral orintrarectal routes. The amount of progastrin or a fragment thereof insuch a composition may be 1 ug to 100 mg. The volume of the compositionadministered may be 100 ul to 2 ml.

The present invention is also directed to a method of inhibitingproliferation of a neoplastic cell, comprising: contacting theneoplastic cell with the composition discussed supra, where thecomposition comprises either an agent that targets progastrin or anagent that targets annexin II or both, such that the contact inhibitsthe growth-inducing activity of progastrin in the cell, therebyinhibiting the proliferation of the neoplastic cell. Generally, thecomposition may block binding of the progastrin to a receptor (AnnexinII) on the cell, may block binding of a protein such as Annexin II thatbinds progastrin, may induce an immune response against progastrin oragainst receptors of progastrin, may downregulate expression ofprogastrin or annexin II at the nucleic acid level or protein level, mayblock internalization of progastrin in the cell or a combinationthereof. Examples of the receptors of progastrin, include but is notlimited to Annexin II, and those proteins that bind progastrinincluding, but not limited to proteins which have molecular mass rangingfrom 40-50 KDa. Additionally, the protein that binds progastrin may bean agonist or an antagonist of progastrin or an inhibitor of progastrinor progastrin binding molecule. The neoplastic cell contacted by thecomposition may be a cancer cell. Examples of cancer cell include but isnot limited to colon cancer cell, breast cancer cell, lung cancer cell,pancreatic cancer cell or any other cancer cell that expresses gastringene or is dependent on gastrin gene products for growth.

The present invention is further directed to a method of treating acancer in an individual, comprising: administering a pharmacologicallyeffective amount of the composition discussed supra to said individual,wherein the composition comprises either an agent that targetsprogastrin or an agent that targets annexin II or both, such that theadministration inhibits gastrin/progastrin-induced proliferation ofcancer cells, thereby treating the cancer in the individual. This methodmay further comprise administering an anticancer drug to the individual.The anticancer drug may be administered prior to, concurrent with orsequentially to the administration. Generally, the anticancer agent maybe a chemotherapeutic agent, an anti-angiogenic agent or ananti-epidermal growth factor agent. The chemotherapeutic agent, theanti-angiogenic agent or the anti-epidermal growth factor agent may beadministered either alone or in combination. Examples of thechemotherapeutic agent may include but are not limited to 5 fluorouracil(5FU), Camptosar (CPT11), Eloxitan (Oxaliplatin), Lavamisol, Leucovorinor irinotecan. The anti-angiogenic agents may include but is not limitedto Avastin and the anti-epidermal growth factor receptor family agentsmay include but is not limited to Erbitux.

Furthermore, the inhibition of proliferation may be due to blocking ofbinding of progastrin to a receptor, such as Annexin II, on the cell,blocking of binding of a protein that binds progastrin, eliciting animmune response against progastrin or receptors of progastrin,downregulating expression of progastrin or annexin II at the nucleicacid or protein level, blocking internalization of progastrin in thecell or a combination thereof. The individual benefiting from thismethod may be one who has high levels of progastrin, is in the primarystages of cancer, has full blown cancer, has tumors induced due to agrowth-inducing effect of progastrin or has previously been subjected tochemotherapy or radiation therapy. Examples of cancer may include but isnot limited to colon cancer, breast cancer, lung cancer, pancreaticcancer or any other cancer that expresses gastrin gene or is dependenton gastrin gene products for growth.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” Some embodiments of the invention mayconsist of or consist essentially of one or more elements, method steps,and/or methods of the invention. It is contemplated that any method orcomposition described herein can be implemented with respect to anyother method or composition described herein.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used herein, the term “contacting” refers to any suitable method ofbringing the composition or antibody described herein into contact witha neoplastic cell. In vitro or ex vivo may be achieved by exposing theabove-mentioned cell to the composition in a suitable medium. For invivo applications, any known method of administration is suitable asdescribed herein

As used herein, the term “pharmacologically effective amount” or“immunologically effective amount” refers to an amount that results inan improvement or remediation of the symptoms of the disease orcondition due to induction of an immune response. Those of skill in theart understand that the effective amount may improve the patient's orsubject's condition, but may not be a complete cure of the diseaseand/or condition.

As used herein, “active immunization” is defined as the administrationof a vaccine to stimulate the host immune system to develop immunityagainst a specific pathogen or toxin.

As used herein, “passive immunization” is defined as the administrationof antibodies to a host to provide immunity against a specific pathogenor toxin.

As used herein, “CpG oligonucleotides” are defined by the presence of anunmethylated CG dinucleotide in a CpG motif.

As used herein, “adjuvant” is defined as a substance which when includedin a vaccine formulation non-specifically enhances the immune responseto an antigen.

The immunogenic composition disclosed herein and the antibody generatedthereof may be administered either alone or in combination with anotheranticancer agent. Such an agent may be administered concurrently orsequentially with the immunogenic composition or antibody disclosedherein. The effect of co-administration with the immunogenic compositionor antibody is to lower the dosage of the anticancer agent normallyrequired that is known to have at least a minimal pharmacological ortherapeutic effect against the disease that is being treated.Concomitantly, toxicity of the anticancer agent to normal cells, tissuesand organs is reduced without reducing, ameliorating, eliminating orotherwise interfering with any cytotoxic, cytostatic, apoptotic or otherkilling or inhibitory therapeutic effect of the drug, compound orantibiotic.

The composition described herein and the anticancer agent may beadministered independently, either systemically or locally, by anymethod standard in the art, for example, subcutaneously, intravenously,parenterally, intraperitoneally, intradermally, intramuscularly,topically, enterally, rectally, nasally, buccally, vaginally or byinhalation spray, by drug pump or contained within transdermal patch oran implant. Dosage formulations of the composition described herein maycomprise conventional non-toxic, physiologically or pharmaceuticallyacceptable carriers or vehicles suitable for the method ofadministration.

The immunogenic composition or antibody described herein and theanticancer agent may be administered independently one or more times toachieve, maintain or improve upon a therapeutic effect. It is wellwithin the skill of an artisan to determine dosage or whether a suitabledosage of either or both of the immunogenic composition or antibody andthe anticancer agent comprises a single administered dose or multipleadministered doses.

As is well known in the art, a specific dose level of such animmunogenic composition or antibody generated thereof for any particularpatient depends upon a variety of factors including the activity of thespecific compound employed, the age, body weight, general health, sex,diet, time of administration, route of administration, rate ofexcretion, drug combination, and the severity of the particular diseaseundergoing therapy. The person responsible for administration willdetermine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

One of skill in the art realizes that the immunologically effectiveamount of the immunogenic composition or the antibody generated thereofcan be the amount that is required to achieve the desired result that isto enhance antibody response against progastrin and/or its receptors.

Administration of the immunogenic composition of the present inventionand the antibody generated thereof to a patient or subject will followgeneral protocols for the administration of therapies used in treatmentof cancer taking into account the toxicity, if any, of the components inthe immunogenic composition, the antibody and/or, in embodiments ofcombination therapy. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described therapy.

As is known to one of skill in the art the immunogenic compositiondescribed herein may be administered along with any of the knownpharmacologically acceptable carriers. Additionally the immunogeniccomposition can be administered via any of the known routes ofadministration such as subcutaneous, intranasal or mucosal. Furthermore,the dosage of the composition to be administered can be determined byperforming experiments as is known to one of skill in the art.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

EXAMPLE 1 Role of Gastrin Gene Expression in Colorectal Carcinogenesis

The stage at which the gastrin gene is re-expressed/over-expressedduring colorectal carcinogenesis is not known in humans. However, thisinformation is important if gastrin gene expression and/or gastrin geneproducts (PG) are to be used as targets for treatment purposes. Hence,the present invention examined the level of expression of gastrin mRNAusing real time quantitative RT-PCR in pinch biopsy of each polyp andsurrounding normal mucosa of patients who had undergone colonoscopy.β-actin and/or 18SRNA was measured as an internal control. All valueswere expressed as a ratio of that in a normal mucosa sample that wasarbitrarily chosen and assigned a value of one.

As shown in FIGS. 2A-2D, ˜75% of normal mucosal samples had negligibleto low levels of gastrin gene expression. On the other hand, only 50%and 40% of hyperplastic (Hp) polyps and adenomas, respectively had lowlevels of gastrin gene expression. A large % of hyperplastic polyps andadenoma specimens (40-50%) and only 35% of normal mucosa were positivefor low to moderate levels of gastrin gene expression. At least 20% ofadenomas were positive for extremely high levels of gastrin geneexpression that were >1000-fold higher than that in the arbitrarilychosen normal control. At least one adenoma sample from a patient wasmeasured to express more than 100,000-fold excess of gastrin genetranscripts compared to the normal control. These results demonstratedfor the first time that there was a great variation in the levels ofgastrin gene expression in polyps. In spite of the small sample size, itbecame evident that on an average the relative levels of gastrin geneexpression were higher in the hyperplastic polyps compared to that inthe normal mucosa and that the levels were many fold higher in theadenomas compared to that in the hyperplastic polyps.

In order to control for possible inter-assay variations, the relativelevels of gastrin gene expression in the polyps were compared to that inHCT-116 cells (wherein the relative level of gastrin gene expression inHCT-116 cells was arbitrarily assigned a value of 1.0). The gastrin geneexpression levels, relative to that measured in HCT-116 cells, ispresented in Table 1, in relation to the location of the polyp and thepathology of the polyp. The data once again demonstrated largevariations, and the standard deviations ranged from 0.54 to 35,604.Therefore, the usual assumption of constant variance and normality willnot apply herein. The relative level of expression of gastrin geneexpression, on an average, was once again confirmed to be more than1000-fold higher than that in HCT-116 cells in the adenomatous polyps(Table 1). The level of gastrin gene expression in the Hp polyps wasalso, on an average, 4 to 5-fold higher than that in HCT-116 cells.These results suggested that PG could potentially play an important rolein the growth of a significant % of Hp polyps. In patients undergoingcolonoscopy, at least one large polyp that was diagnosed as anadenocarcinoma (AdCA) (Table 1) was found to express relatively lowlevels of gastrin RNA. The gastrin RNA expression will be examined inmany more AdCA samples in future.

It was interesting to note that the relative expression of gastrin RNAwas many fold higher in polyps removed from either the proximal or thetransverse (sigmoid) portions of the colon, while the levels appeared tobe much lower in polyps from the descending colon and the rectum (Table1). Equally surprising was the finding that ˜60% of polyps were obtainedfrom the ascending (proximal) colon and another 20% were obtained fromthe transverse section of the colon (Table 1). In animal studies, apredominance of pre-neoplastic lesions was reported in the proximalcolon in mice over-expressing PG, which appears to resemble theprevalence of proximal polyps in humans as well (15-17). It is thuspossible that the etiology and risk factors dictating the prevalence ofproximal colon carcinogenesis (in terms of Hp and Ad growths) may besimilar in humans and in animal models over-expressing PG.

To confirm the rather unexpected finding that at least 50% of Hp polypsexpressed significant levels of gastrin RNA (FIGS. 2B, 2D),paraffin-embedded sections of representative polyps were examinedimmunocytochemically with specific anti-PG-Abs using previouslydescribed procedures (1, 15-17). A significant % ofhyperplastic/dysplastic polyps stained variably for PG (FIG. 3A).Sections from at least one paraffin-embedded AdCA were also processedimmunocytochemically with anti-PG-Ab, and was found to stain heavily forPG (FIG. 3A). Immunofluorescent staining confirmed a relative increasein the expression of progastrin associated with disease progression,wherein relative levels of PG were highest in adenocarcinomas followedby adenomas, with negligible expression in normal colons (FIG. 3B, TableII).

EXAMPLE 2 Growth Effects of Autocrine Progastrin (PG)

Since PG is the major form of gastrin expressed by primary colon cancersand colon cancer cell lines, the effect of specific antibodies on thegrowth of gastrin-dependent human colon cancer cells was examined.Specific anti-PG-AB at concentrations of 1:500 abolished the growth ofgastrin-dependent cell lines (Colo-320, DLD-1, HCT-116) to basal,non-stimulated levels while anti-GG-Ab was much less effective andanti-G17-Ab was ineffective. Representative data from HCT-116 cells arepresented in FIGS. 5A-5B. Thus, it was confirmed that colorectal cancersdo not express amidated gastrins.

EXAMPLE 3 Development of a rhPG Specific Vaccine

Since autocrine and endocrine PG might be playing an important role incolon carcinogenesis, the possibility of using an rhPG fusion protein asan immunogen for treatment purposes was examined. Briefly, the rhPGfusion protein (FP) was used as an immunogen in Balb/C, FVB/N andFABP-PG mice. The immunogen generated a strong response and very hightiters of specific anti-hPG-Abs were measured within 4-8 weeks ofvaccination. Data for Balb/c mice is shown in FIG. 6A. Mouse coloncancer (CA) cells are derived from Balb/C tumors and grow subcutaneouslyin Balb/C mice (4). The effect of high titers of anti-hPG-Abs inimmunized mice was examined against the growth of CA cells in Balb/Cmice (FIG. 6A). Mouse PG shares ˜80% homology at the Cterminal end, and<65% homology at the N-terminal end, with hPG. CA tumor weights in micetreated with hPG-immunogen were reduced 15-30% compared to that incontrol mice. Additionally, in one mouse that demonstrated the highestAb titer, tumors were not only the smallest, but also necrotic (FIG.6A). Similarly, FVB/N mice, immunized against hPG-FP, were treated withAOM to examine if anti-PG-Abs can reduce the risk of coloncarcinogenesis in these mice. The total number of tumors (FIG. 6B) andtumor burden/mouse (FIG. 6C) were significantly (p<0.05) reduced in miceimmunized against hPG peptide compared to mice immunized with theadjuvant alone.

Aberrant crypt forci (ACFs) are pre-neoplastic markers of coloncarcinogenesis and represent dysplastic growth of colonic crypts, whichis the earliest stage of pre-cancerous changes in the colonic mucosa asdescribed in detail previously (15). In a transgenic mouse model (PS21,Fabp-PG), which overexpressed human progastrin peptide in the coloniccrypts (17), it was demonstrated that the mice were at very high riskfor developing ACF and adenomas and adenocarcinomas in response to acarcinogen, azoxymethane (17). These mice were immunized with eitherthioredoxin, or the progastrin peptide alone or the fusion protein (FP)which contained thioredoxin and progastrin.

Briefly, transgenic mice expressing human progastrin (PS21) and wildtype mice were divided into 3 groups and immunized with (a) recombinantthioredoxin, (b) a recombinant fusion protein where human progastrin wasfused with thioredoxin and recombinant progastrin, all proteins wereexpressed in E. coli and purified from cell extract. Each protein wasemulsified using CFA/IFA (Sigma) as an adjuvant. The mice that receivedvaccine emulsified using CFA on day 1 followed by two subsequentimmunization using IFA on day 21 and day 42. All injections wereadministered subcutaneously. Mice were bled and serum prepared toestimate the titers once a week for 3 weeks, 15 days after the finalimmunization. Mice were sacrificed after receiving AOM injection and thecolons were excised and fixed. The ACFs were then counted.

FIG. 7A shows the number of aberrant crypt foci (ACF)/PS21 transgenicmice or wild type mouse and FIG. 7B shows the total number of aberrantcrypt foci/mouse colon. The antibody tires in serum were determined bysolid phase ELISA using limiting dilution method. Briefly, 96 wells PVCELISA plates (Falcon) were coated with 3 different recombinant proteinexpressed in E. coli that were used to vaccinate mice i.e. recombinantthioredoxin, Thioredoxin-PG fusion protein and recombinant PG.Recombinant proteins were dissolved in carbonate buffer pH 9.0 andcoated at a concentration of 50 μg/50 μl/well. The plates were incubatedovernight at 4° C. Following day the plates were flipped and the wellwere blocked with 100 μl 2% non fat dry milk (NFDM) and plated wereincubated at 37° C. for 3-4 hours. The plated were then washed 3 timeswith phosphate buffered saline containing 0.5% tween-20 (PBST). Thecoated plated were then loaded with 100 μl PBST and stored at −20° C.till further use. To determine tires the serum was diluted in PBST and50 μl of diluted was loaded into the well and further diluted seriallyusing double dilution. The plated were incubated at 4° C. overnight. Theplates were then washed 4 times with PBST and 50 μl of rabbit anti-mouseIgG from Biorad (diluted 1:3000) was added to each well and incubatedfor 3 hrs at 37° C. the plates were then washed and incubated withorthophenlyamine-HCl (8 mg/10 ml) containing 100 μl of H₂O₂ at 37° C.for 10 min. The reaction was stopped by 50 μl of 5N H₂SO₄. The ELISAplated were them read in plate reader at 490 nm. The titer value wasdefined as the inverse of highest dilution at which the OD was at leasttwice higher than the control preimmune serum.

Mice that were immunized with the PG peptide alone demonstrated muchlower levels of anti-PG-antibody titers, in both PS21 and WT mice, whilemice immunized with the fusion protein demonstrated 2 fold to muchhigher titers of the anti-PG antibodies, especially in the WT mice(FIGS. 7C-7D). The generation of PG antibodies (while 2 fold higher inPS21 mice injected with FP compared to PS21 mice injected with PGalone), was relatively attenuated in FP injected PS21 versus WT mice,which may reflect high levels of hPG since birth in PS21 mice, which isprobably recognized as a self antigen. There is thus a good possibilitythat in mice/humans vaccinated against FP, the anti PG titres will bevery high, since PG is not secreted at very high levels in wildtypeanimals. Mice immunized with thioredoxin demonstrated significant titersof anti-thioredoxin-antibodies in all the mouse groups (FIG. 7E). Thedevelopment of ACF in response to AOM was significantly enhanced in PS21mice immunized with either PG alone or with thioredoxin (FIGS. 7A-7B),as measured previously (17). However, mice immunized with the fusionprotein (FP) demonstrated a complete attenuation of the development ofACF in response to over-expressed PG, and the number of ACFs in the FPimmunized PS21 mice reverted to control levels measured in wild typemice (FIGS. 7A-7B), providing strong evidence that colon carcinogenesisin response to progastrin can be effectively attenuated by immunizingwith the fusion protein. Thus, the results presented herein indicatedthat the strategy of using progastrin peptides as immunogens would beextremely effective in neutralizing biological effects of PG.

EXAMPLE 4 Binding Affinity and Relative Binding Affinity (RBA) ofPeptides for ¹²⁵I rhPG Binding Sites: Identification of PG Receptors asAnnexin II

The full-length recombinant human PG₁₋₈₀ (rhPG) was generated in an E.coli expression system and purified and confirmed as describedpreviously (11). rhPG was radio labeled with Na¹²⁵I (Amersham, Chicago,Ill.), and the intact ¹²⁵I-rhPG purified by HPLC as described previously(11). For these experiments, AR42J cells were expanded in vitro in 175mm flasks and grown to subconfluence in the growth medium containing 10%FCS. All binding assays were performed 36-48h after seeding the cells inculture medium containing 10% FCS and 2% glutamine. Before the start ofthe binding assays, the cells in culture were washed with Hanks Balancedsalt solution (HBSS) (GIBCO) containing 0.1% BSA and 25 mM HEPES (Sigma)and scraped with a rubber policeman into conical tissue culturepolystyrene tubes. Cells were centrifuged at 500g for 5 min andresuspended in HBSS at a concentration of 2×10⁶ cells/ml. Aliquots (˜1.0ml) of suspended cells in polystyrene tubes were used in the bindingassays. Binding assays were conducted as described previously (6,11).For purposes of determining binding affinity, a multipoint (7-12 points)saturation analysis was performed by using increasing concentrations(0.03-1.0 nM) of ¹²⁵I-rhPG without (total binding) or with (non-specificbinding) 1,000-fold excess of radio inert rhPG. The binding data wasanalyzed by a Scatchard plot as described previously (6). Binding assayswere performed at 37° C. for 30-60 min at pH 6.5 (optimal for binding).

In order to define the RBA of various peptides for the PG binding siteson the cells, cells in suspension were incubated with 1.0 nM ¹²⁵I-rhPGin the presence or absence of increasing concentrations (0.1 nM-10.0 μM)of either the homologous or heterologous peptide. Non-specific bindingwas determined in the presence of 1000× excess of the non-labeledhomologous peptide. At the end of the incubation, the cells werepelleted and washed twice with 1 ml of fresh ice-cold HBSS plus 0.1%BSA. Cell pellets were counted for ¹²⁵I in a gamma counter with ˜70%efficiency for ¹²⁵I. The RBA of gastrin-like peptides and/or competingpeptides for binding the specific binding sites for rhPG were determinedfrom a log-dose inhibition of specific binding of ¹²⁵I-rhPG by variouspeptides as described previously (6). In a few experiments, ¹²⁵I-BoltonHunter-CCK-8 (Amersham Biosciences, Piscataway, N.J.) was used as theradiolabeled ligand and the RBA of gastrin like peptides for displacingthe binding of ¹²⁵I-BH-CCK8 to AR42J cells examined by our publishedmethods (6).

It is by now well known that gastrin and progastrin peptides exertbiological effects on target cells via novel binding sites that areseparate from CCK₂R (2). Since CCK₂R are expressed at highconcentrations on AR42J cells (6), the relative binding affinity ofgastrin-like peptides (PG, G17 and CCK) was examined for the highaffinity CCK receptors and the high affinity PG binding receptors. Asshown in FIGS. 8A-8C, PG dose-dependently displaced the binding ofradio-labeled rhPG (FIG. 8A, 8B), but was largely ineffective indisplacing the binding of radio-labeled ¹²⁵I-BH-CCK8 to CCK₂R on theAR42J cells (FIG. 8C).

On the other hand, CCK and G17, both of which bind CCK₂R with highaffinity (2), demonstrated a negligible binding affinity for the PGbinding sites (FIG. 8A, 8B), but completely displaced the binding of¹²⁵I BH-CCK8 (FIG. 8C). These results confirm that PG does not bindCCK₂R, but binds novel receptors with high affinity, which was recentlyconfirmed to be annexin-II (14). Annexin-II has been shown to play animportant role in mediating growth factor effects of PG on colon cancercells (14), intestinal epithelial cells (FIGS. 4J-4L) and pancreaticcancer cells (FIG. 4M) (9, 13-14). Annexin II demonstrated avid bindingto progastrin in vitro and in vivo (FIGS. 4B-4I), demonstrated a strongco-localization with progastrin in situ (FIG. 4I) and was required forbinding and internalization of the progastrin peptide (FIG. 4N).

EXAMPLE 5 Relative Expression of CCK2R in Hyperplastic and AdenomatousPolyps and in Adenocarcinoma Samples

Using Real Time quantitative RT-PCR, the relative levels of CCK₂receptors in polyp and tumor specimens were also measured. As can beseen in FIGS. 9B-9C, low to negligible levels of CCK2R were measured inthe normal mucosa of patients from whom polyps were obtained at the timeof colonoscopy. None of the hyperplastic polyps and adenomas expresseddetectable levels of CCK2R (FIG. 9B). In the frozen adenocarcinomasamples, and in the majority of the surrounding normal colonic mucosalsamples from these patients, no CCK2R RNA was measured (FIG. 9D). Onlyone normal mucosal sample (obtained from the tumor bank from thecorresponding CRC patients) was positive for CCK2R RNA (FIGS. 9B-9C).The presence of low levels of CCK2R in the normal colonic mucosa, andalmost complete absence of CCK2R expression in the colonic growths(including hyperplastic growths) suggests that CCK2R does not play arole in colon carcinogenesis. If anything, the results presented hereinsuggest that down-regulation of CCK2R may be required or acquired duringthe process of initiation and progression of the colorectal cancerdisease.

EXAMPLE 6 Sequences of the Vectors and Nucleic Acid Encoding thePeptides and Amino Acid Sequences of the Peptides Discussed Herein

The complete description of the E. coli vector that was used forexpressing the fusion protein containing bacterial thioredoxin and humanprogastrin is presented in FIGS. 10A-10B. The description of the vectoris provided as text in FIG. 10B in some detail, and is self-explanatory.The hGastrin cDNA sequence is shown in SEQ ID NO: 1. The changes thatwere made in the hGastrin cDNA sequence for purpose of codonoptimization (SEQ ID NO: 2) in order to obtain efficient expression ofthe PG peptide from the E. coli system used, is provided in FIGS.10C-10F. Once again the information is self-explanatory. Finally the 80amino acid sequence of the human PG peptide (SEQ ID NO: 3) thusexpressed and confirmed by mass spectrophotometery and by sequencing ispresented in FIG. 10G. The symbol for each amino acid is shown.

The following references were cited herein:

-   1. Singh et al, Curr Opin Gastroentrol 2000a, 16:68-77.-   2. Rengifo-Cam and Singh, Curr Pharmaceutical Design 2004,    10:2345-2358.-   3. Siddheshwar et al, Gut 2001, 48:47-52.-   4. Singh et al, Cancer Res 1996, 56:4111-4115.-   5. Seva et al., Science 1994, 265:410-412.-   6. Singh et al, J Biol Chem 1995, 270:8429-8438.-   7. Rosenberg et al., Nat Med 2004, 10(9):909-915.-   8. He and Marshall, Expert review of anticancer therapy 2006,    6(4):487-492.-   9. Singh et al, Oncogene 2007, 26(3):425-440.-   10. Brown et al, Endocrinology 2003, 144:201-211.-   11. Singh et al, Am J Physiol Gastrointest Liver Physiol 2003,    284:G328-G339.-   12. Wu et al, Am J Physiol Gastrointest Liver Physiol 2003,    285:G1097-G1110.-   13. Rengifo-Cam et al, Cancer Research 2007, 67(15):7266-74.-   14. Singh, Cancer Letters, 2007 252:19-35.-   15. Singh et al, Am J Physiol Gastrointest Liver Physiol 2000b, 278:    G390-G399.-   16. Singh et al, Gastroenterology 2000c, 119: 162-171.-   17. Cobb et al, Cancer 2004, 100:6 1311-1323.-   18. Singh and Cobb, Gastrin in the New Millennium 2004, 319-327.-   19. Olivo and Wargovich, In Vivo 1998, 12: 159-166.-   20. Takayama et al, N Engl J Med 1998, 339: 1277-1284.

What is claimed is:
 1. A method of treating colorectal cancer in a humancomprising: administering to a human subject suffering from colorectalcancer a composition comprising a pharmaceutically effective amount ofan antibody that specifically binds human progastrin, wherein theantibody blocks binding of human progastrin to an Annexin II receptor,wherein the antibody inhibits the growth-inducing activity of progastrinin the cell, and wherein the antibody is monoclonal or polyclonal madeby immunizing with full length recombinant human progastrin.
 2. Themethod according to claim 1, further comprising administering ananti-cancer agent.
 3. The method of claim 2, wherein the anti-canceragent is administered before the antibody.
 4. The method of claim 2,wherein the anti-cancer agent is administered concurrent with, orsequentially after the antibody.
 5. The method of claim 2, wherein theanti-cancer agent is a chemotherapeutic agent.
 6. A method of treating acolorectal cancer, a pancreatic cancer, or a gastrointestinal cancer ina human comprising: administering to a human subject suffering fromcolorectal cancer, pancreatic cancer, or gastrointestinal cancer acomposition comprising a pharmaceutically effective amount of an agenttargeting human progastrin, wherein the agent is an antibody thatspecifically binds human progastrin or a fragment thereof, wherein theantibody results in blocking binding of human progastrin to an AnnexinII receptor, wherein the antibody inhibits the growth-inducing activityof progastrin in the cell, and wherein the antibody is monoclonal orpolyclonal made by immunizing with full length recombinant humanprogastrin.
 7. The method of claim 6, wherein the subject is sufferingfrom colorectal cancer.
 8. The method of claim 6, wherein the subject issuffering from pancreatic cancer.
 9. The method of claim 6, wherein thesubject is suffering from gastrointestinal cancer.
 10. The method ofclaim 6, further comprising administering an anti-cancer agent.
 11. Themethod of claim 10, wherein the anti-cancer agent is administered beforethe antibody.
 12. The method of claim 10, wherein the anti-cancer agentis administered concurrent with, or sequentially after the antibody. 13.The method of claim 10, wherein the anti-cancer agent is achemotherapeutic agent.